Brain Proteomics in Smith-Lemli-Opitz Syndrome (SLOS). This investigation uses mouse models for human genetic diseases involving defects in cholesterol biosynthesis. 2D gel-based proteomic studies of wild type and knockout mice (Dhcr7 and Sc5d) brain tissue have been led to the determination of 64 proteins with expression differences exceeding 1.5-fold as determined from image analysis of triplicate 2D gels. For protein identification, at least 3 peptides were matched with greater than 95% confidence, and total amino acid sequence coverage ranged from 14% to 74% of the total protein. HMG CoA synthase is an example of a differentially expressed protein observed in brain extracts. HMG CoA synthase in brains of knockout animals was significantly increased relative to wild type, with a ratio of control to knockout equal to 1:1.55 for Dhcr7, and 1:1.75 for Sc5d. Since HMG CoA synthase is the first step in the cholesterol biosynthetic pathway, a decrease in brain cholesterol levels would be expected to increase the activity/expression of this enzyme, which is what was observed for both knockouts. In addition to changes in the level of protein expression, this approach also detected changes in protein post-translational modifications in brain proteins from knockout mice. These modifications were detected from a shift in protein mobility on the gel. An example of this change was observed for cofilin 1. Two gel spots were observed to contain cofilin 1, and the spot migrating with a more acidic isoelectric point was observed to have an increased intensity in knockout mice relative to wild type controls. Western Blots, using an anti-phospho cofilin 1 antibody, confirmed the increase in cofilin 1 phosphorylation. Another example of a shift in protein migration was observed for the Rab7 protein. Rab 7 is a member of a large family of GTP-binding proteins. Many of the proteins in this family are modified at the C-terminal by prenylation at C-terminal cysteine residues. Protein prenylation is a side branch in the biochemical pathway for cholesterol biosynthesis, and alternations in cholesterol biosynthesis could also result in changing the levels of protein prenylation. Prenylation is known to shift the mobility of at least some GTP-binding proteins, and the data suggest that a change in Rab7 prenylation may result in the observed mobility shift. However, a direct determination of Rab7 prenylation was not performed here, and other post-translational modifications may also be involved. Lipid Quantification in Serum. We have continued developing methodology to quantify cardiolipins in human serum by mass spectrometry. This effort is in association with a clinical study underway in the Institute to evaluate the effects of antibiotic treatment of pregnant women colonized with the Group B streptococcal (GBS) organism. The hypothesis of the study is that the typical peri-natal penicillin treatment gives rise to a large increase of circulating cardiolipins in the infant which then leads to respiratory distress. It has been demonstrated in other studies in newborn sheep that the GBS organisms secrete a specific cell wall membrane cardiolipin with penicillin treatment and that this substance causes respiratory distress at levels corresponding to the injection of about 100 pmole/mL in serum;note that this concentration falls rapidly with a half life of a few minutes. It is not known whether the respiratory distress observed in a fraction of infants born to GBS colonized mothers is a result of a similar effect, or perhaps by a related effect caused by a release of endogenous cardiolipins stimulated by the bacterial death. The analytical approach involves the addition of an internal standard to a serum sample, extractions by a combination of liquid-liquid and solid phase, followed by an LC-MS analysis that incorporates an extraction/recovery standard to monitor system quality control. We have shown that cardiolipin can be extracted from serum with approximately 90% efficiency. We have further shown that normal adult levels of (18:2)4 cardiolipins are present in serum at levels <10 fmole/uL, approximately 1000-fold lower than found by earlier, less accurate measurements. With this base line information, we are seeking to establish normal serum levels in cord blood of mothers known to be uncolonized by GBS and will then determine levels in infected individuals. Quantification of Histone de-Methylation. Histone methylation regulates chromatin structure, transcription, and epigenetic state of the cell. Histone methylation is dynamically regulated by histone methylases and demethylases which mediate demethylation of di- and monomethylated histones. It has been unclear whether demethylases exist that reverse lysine trimethylation. A putative histone demethylase, KDM3B that was identified in earlier investigations, is being investigated to probe the specificity and extent of its demethylation capability using a model peptide synthesized with a lysine residue containing 1, 2 or 3 methylations. The methylated peptide, ARTKQTARKSTGGKAPRKQLAGGK-biotin, is incubated with either immuno-precipitated (IP) or recombinant KDM3B and the ratio of de-methylated product to substrate is determined by MALDI/TOF. Replicate mass spectral measurements of the peptide substrate from control and enzyme experiments, both with and without the putative co-factor RanQ69L-GTP, are corrected for background levels and the fractional de-methylation calculated. Initial studies with recombinant KDM3B showed no activity, while the IP enzyme showed activity for de-methylation of 1-Me, removal of both methyl groups from 2-Me and no activity with the 3-Me substrate.